Project Summary/Abstract Candidate. I have a profound interest in understanding the genetic and epigenetic mechanisms that control cell state, and have built upon that interest during my research career. As an undergraduate researcher in the laboratory of Dr. Joanne Wiley at Hofstra University, I studied bacterial sporulation and antibiotic production using genetics and scanning electron microscopy, resulting in authorship on a peer-reviewed manuscript. As a Ph.D. candidate in the laboratory of Dr. Rudolf Jaenisch at the Massachusetts Institute of Technology, I worked on several of projects which resulted in authorship of peer-reviewed manuscripts, related to epigenetics, factor- mediated reprogramming, and transdifferentiation. In my main thesis work, I used genomics, genetics, molecular biology, and tissue culture techniques to study cell fate transitions in the development of the mammalian nervous system, resulting in a first-author publication (Lodato et al., PLoS Genetics, 2013, PMID 23437007). In the first part of my postdoctoral fellowship, in the lab of Dr. Christopher Walsh of Boston Children?s Hospital and Harvard Medical School, I built upon all these previous experiences, in particular in using genomics to understand principles of neurobiology, to study somatic mutations in the normal human brain using cutting-edge single-cell whole genome sequencing (WGS) technologies, resulting in a first-author manuscript (Lodato et al., Science, 2015, PMID 26430121). This paper was the first published work to compare the whole-genome rates and characteristics of somatic mutations across normal human individuals, and the expertise I gained developing that technology has prepared me thoroughly to now compare the rates and characteristics of somatic mutations between age groups and in human age-related diseases. The K99/R00 Pathway to Independence Award is the perfect mechanism to help propel me to my scientific and career goals. Under this award, I will 1) Execute a research plan to uncover the role of somatic mutation in aging and in age-related diseases 2) Gain scientific skills and refine my understanding of key concepts in the fields of aging and bioinformatics 3) Undertake career development activities under the supervision of my mentor, co-mentor, and other faculty advisors committed to my successful transition from postdoctoral fellow to independent investigator. Environment. The proposed Research and Training plans will take place in the laboratory of Christopher A. Walsh, M.D., Ph.D., within Boston Children?s Hospital (BCH) and Harvard Medical School (HMS). These institutions comprise a strong, well-established research community, and are committed to the success of the aims and goals proposed in this application. Dr. Walsh has a strong track record of training postdoctoral fellows, many of his former trainees now holding tenured or tenure-track faculty positions at academic institutions. This success stems from the rigorous and supportive environment in the lab, including frequent lab meetings and one-on-one interactions with Dr. Walsh. Scientifically, the lab is supplied with virtually all reagents and equipment needed for the proposed research, and as a part of the greater BCH and HMS community any additional equipment are available within walking distance. The BCH/HMS community is also an asset to the career development aspects of this proposal, providing easy access to potential collaborators and frequent seminars hosting internal and external speakers. As part of this community, and I have secured a co-mentor (Dr. Peter Park, HMS), and two additional faculty advisors (Drs. Bruce Yanker, HMS, and Emanuela Gussoni, BCH/HMS), all committed to my success. Finally, the BCH/HMS environment provides myriad formal career development seminars and workshops, which I will take part in to aid my transition to independence. Research. Advanced age is a major risk factor for human diseases in all organ systems, yet how the phenomenon of aging affects such a wide spectrum of tissues is unknown. All tissues rely on the integrity of the genome to function properly, and one attractive and long-standing hypothesis is that the gradual accumulation of DNA damage might be causal in aging. Proof of this notion has remained elusive in the brain, since standard DNA-sequencing experiments are ill-suited to detect somatic mutations which might mark only a few cells, or even a single cell, in a sample comprised of millions. I have pioneered the use of single-cell, whole-genome sequencing technology to compare rates, characteristics, and consequences of somatic mutations across human brains, and will use this technology as a part of this award to achieve three Aims: 1) Determine whether aging is associated with an accumulation of mutations in the human brain and define patterns of somatic mutation during aging, 2) Single-cell sequencing in progeroid diseases associated with DNA-damage response, and 3) Examination of the somatic mutation rate in Alzheimer?s disease. Thus, this proposal represents a comprehensive body of work that will elucidate the role of somatic mutation in during aging, in accelerated aging, and in age-associated disease in the human brain.